1. Field of the Invention
The present invention relates to cell culturing devices, and in particular, it relates to cell culturing devices having a plurality of hollow fiber membranes that efficiently and selectively transfer oxygen, nutrients and other chemical stimuli and remove waste products to grow and maintain cells in vitro at high cell densities to provide high yields of product formation per unit reactor volume.
2. Description of the Prior Art
Cell culture devices for culturing cells in vitro having a shell with a plurality of hollow fiber membranes have been known for quite some time. Medium containing oxygen, nutrients and other chemical stimuli is transported through the lumen of the hollow fiber membranes and undergoes a pressure drop resulting in an outward radial convective flow at the entry port of the device and an inward flow at the exit port of the device. Cells are grown in the fluid space between the fibers and the shell wall.
Hollow fiber culture devices have been proven to be ideal for the maintenance of many types of cells at high densities in vitro. The mass transfer characteristics of hollow fiber culture devices provide an efficient means of delivering nutrients and removing waste products from a culture. The semi-porous hollow fiber membranes can be selected with various pore sizes. With proper pore size selection, the cellular product can be maintained on the outside of the fibers, while waste products and contaminating proteins will pass through the membrane pores into the lumen of the hollow fibers where they can be subsequently removed from the culture.
To economically produce cell-derived products in a hollow fiber culture device, large numbers of the cells must be maintained viable in optimal culture conditions for product formation over long periods of time. Prior art hollow fiber culture devices have many limitations that prevent their use in the economical production of cell-derived products in commercial quantities. These limitations include: (1) formation of gradients in the cell compartment; (2) inability to directly monitor and control cellular environment; (3) lack of fluid movement in cell compartment leads to microenvironment formation around cells; (4) fibers are not equidistant apart in culture device leading to anoxic or dead spaces; (5) efficient mass transfer becomes difficult at high cell densities; and (6) the pressure drop across the device increases as the device is scaled up, increasing the problems cited above, thus limiting scaleability. The purpose of the present invention is to overcome these limitations, making it possible to utilize a hollow fiber culture device for the economical production of cell-derived products. The following explains each of the prior art limitations in more detail.
1. Formation of Gradients in the Cell Compartment.
Formation of gradients of nutrients, pH, O.sub.2, CO.sub.2, lactic acid, ammonia and other components of the culture media in the cell compartment is a common problem in hollow fiber culture devices. The gradients form due to the manner in which nutrient media flows through the device. Nutrients are more available to the cells near the inlet port due to the outward flow from the hollow fibers. As media flows to the outlet, metabolic waste products, such as lactic acid and ammonia, accumulate in the cell compartment undesirably affecting cell viability.
2. Inability to Monitor and Control Cell Compartment.
Because of the nature of prior art hollow fiber cartridges, it is not possible to incorporate monitoring devices into the cell compartment. Thus, maintenance of optimal culture conditions is very difficult. The only control over the cell compartment environment is by diffusion of products from the lumen of the hollow fibers to the cell compartment.
3. Formation of Microenvironments.
Very little fluid motion occurs in the cell compartment of prior art hollow fiber culture devices. This leads to microenvironments forming around quickly metabolizing cells, adversely affecting other cells in the device by altering pH.
4. Formation of Anoxic Pockets.
Hollow fibers are packed into cartridges. Usually the fibers are not equidistant apart and thus, some cells are farther away from the nutrient source than others. This leads to pockets where cells will die due to lack of oxygen or failure of nutrients to reach the cells by diffusion.
5. Mass Transfer Limitations.
As the cells grow to high cell densities, nutrients must diffuse through greater layers of cells. Mass transfer by diffusion is limited, limiting the number of cells that can be maintained in the culture device.
6. Pressure Drop.
Hollow fiber culture devices perfuse cells in the cell compartment and remove waste products due to the forces set up by the pressure drop that occurs across the cartridge. This pressure drop becomes greater as the length of the fibers are increased. This pressure drop also creates the gradient problems described above. Thus, the length of the fibers is limited by the pressure drop which enhances the gradient problem.
Some examples of prior art cell culturing devices are described in the following patents:
______________________________________ Inventor U.S. Pat. No. ______________________________________ Matsumura 3,734,851 Knazek et al 3,821,087 Knazek et al 3,883,393 Osborne et al 3,911,140 Delente 3,997,396 Feder et al 4,087,327 Knazek et al 4,184,922 Knazek et al 4,200,689 Feder et al 4,201,845 Knazek et al 4,206,015 Knazek et al 4,220,725 Chick et al 4,242,460 Yoshida et al 4,391,912 Hsei 4,396,510 Michaels et al 4,440,853 Michaels et al 4,442,206 ______________________________________